Regarding Malignant Tumor:
A malignant tumor (cancer) is the first leading cause of death in Japan and the number of patients is increasing every year, and the development of a drug and a therapeutic method having high efficacy and safety is strongly desired. As the cause of forming a malignant tumor, there is a mutation of DNA by radiation, ultraviolet rays and various carcinogenic substances. Studies on malignant tumors have been focused on molecular biological identification of these genetic changes. As a result, it is considered that tumorigenic transformation is induced by accumulation of a large number of mutations and the like. It has been shown by a cell line model and the like that some decisive mutations directly connected with the tumorigenic transformation. Regarding leukemia as one of the objective diseases of the invention, many chromosomal abnormalities have been identified and classified. In many of the case, translocation of chromosome is found and the some genes associated with chromosomal translocation have already been identified in principle chromosomal translocations. By analyses of functions of the translocation related genes, a case has been found that these genes are concerned in the onset of leukemia.
Regarding Cancer Stem Cell:
On the other hand, a so-called cancer stem cell hypothesis has been proposed for a long time from the viewpoint of cell biology, stating that stem cell is the origin of a malignant tumor similar to the normal tissue. The stem cell is defined as a cell having autonomous replication ability and pluripotency and generally divided roughly into totipotency stem cell and tissue stem cell. Tissue stem cells are originated from specific tissues and organs such as of blood system, liver, nerve system and the like and present at an extremely low frequency. Among them, hematopoietic stem cell has been studied most frequently. It has been reported that a hematopoietic system can be reconstructed over a long period of time by transplanting one hematopoietic stem cell into a mouse in which the hematopoietic system was destructed by a lethal dose of irradiation (Non-patent Document 1). Different from the normal stem cell, studies on cancer stem cells have been delayed for a prolonged period of time since their true nature could not been found. However, a cancer stem cell has been identified for the first time in acute myeloid leukemia, in 1997 by Dick et al. Thereafter, the presence of cancer stem cells has been reported in various malignant tumors. In summing up, cancer stem cells are present at a frequency of several % or less of the whole tumor and rare as well as normal stem cells. It is considered that the remaining cells which form the tumor are tumor precursor cells in which proliferation ability is limited or tumor cells.
By these reports, it was shown that hierarchy is present even in tumor similar to the normal tissue, and the cancer stem cell residing at this peak (origin) has strong tumor forming ability.
Characteristics and Therapeutic Problems of Cancer Stem Cells:
In summing up many reports, it is considered that cancer stem cells are maintaining various characteristics possessed by the normal stem cells. Examples of similarities include the rarity of the cells, a microenvironment (niche) in which the cell exists, expression of a multiple drug resistance gene, cell cycle arrest, and the like.
Particularly, the characteristics that they express a group of multiple drug resistance genes and are at the interphase of cell cycle similar to the normal stem cells could become a therapeutically great problem. A multiple drug resistance gene BCRP is a pump which impairs the drug efficacy by eliminating various antitumor agents into outside of cells, and a method for collecting stem cells making use of the activity has been reported (Non-patent Document 2). In addition, their presence at the interphase of cell cycle under a state of “arresting” (Non-patent Document 3) is causing reduction of sensitivity for many antitumor agents and radiations which targets the quick cell growth of cancer (Non-patent Documents 4 and 5).
Based on the above characteristics, it is considered that the cancer stem cell which exhibiting resistance to the therapy is a cause of tumor regeneration.
Regarding Molecular Target Drug
Three main courses of the treatment of a malignant tumor include of antitumor agent therapy, radiation therapy and surgical excision. The blood tumor is limited to the antitumor agent therapy and radiation therapy, and as described in the above, the cancer stem cell can have a resistance to these treatments. Another problem is that side effects are large since these two treatments affect the entire body. It is a molecular target drug that is expected as a resolving means for this problem. It has a possibility to reduce side effects by exhibiting its drug efficacy only in the cell expressing the target molecule.
Examples of typical drugs of the molecular target drug in the field of blood diseases include imatinib and rituximab. Imatinib targets at a leukemia-causing factor called Bcr-Abl produced by a chromosomal abnormality (Philadelphia chromosome) which is observed in 95% of CML patients. This is a low molecular weight drug which induces suicide of leukemia cell by inhibiting function of Bcr-Abl. Rituximab is a therapeutic antibody which recognizes CD20 as a surface molecule on a B cell and has an antitumor effect on a malignant tumor of B cell (non-Hodgkin lymphoma and the like). On the other hand, molecular target drugs for AML are few, and there is only an agent gemtuzumab.ozogamicin (Mylotarg) in which an antibiotic calicheamicin is linked to a monoclonal antibody for CD33 known as an AML cell surface antigen. However, it is the present situation that the use of Mylotarg is limited because of its strong toxicity which is considered to be derived from calicheamicin in addition to the problem that therapeutic range is narrow. Based on the above, it can be said that discovery of a new target gene and development of a therapeutic agent for this are important inventions which directly lead to the possibility of therapy and expansion of the choices of therapy.
As the embodiment of molecular target drugs, various substances have been studied and developed such as a therapeutic antibody and a low molecular weight drug, as well as a peptide drug, a biological protein preparation such as cytokine, an siRNA, aptamer and the like. When an antibody is used as a therapeutic agent, due to its specificity, it is useful in treating pathological conditions in which the disordered cell expresses a specific antigen. The antibody binds to a protein expressing on the cell surface as its antigen and effectively acts upon the bound cell. The antibody has a characteristic of long blood half life and high specificity for its antigen and is also markedly useful as an antitumor agent. For example, when an antibody targets at a tumor-specific antigen, it can be expected that the administered antibody accumulates into the tumor and thereby attacks the tumor cell via complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). In addition, by binding a radioactive substance, a cytotoxic substance and the like to an antibody, it becomes possible to transfer an agent efficiently to the tumor part and thereby to allow to act thereon. At the same time, it can decrease the amount of the reached agent to non-specific other tissues and reduction of side effects can also be expected. Inhibition of tumor growth or regression of tumor can be expected by administering an antibody having agonistic activity when a tumor-specific antigen has an activity to induce cell death, or by administering an antibody having neutralization activity when a tumor-specific antigen relates to in the growth and survival of cells. Due to the above characteristics, it is considered that antibodies are suited in applying as antitumor agents.
Regarding Therapeutic Antibodies:
In the original antibody preparation, a mouse was used as the animal to be immunized. However, use of mouse antibodies as drugs is limited due to a large number of reasons. A mouse antibody which can be recognized as a foreign substances in the human body can induce so-called “human anti-mouse antibody” namely “HAMA” response (Non-patent Document 6). Further, the Fc region of mouse antibody is not effective for the attack on disease cells via human complement or human immune cells.
As one of the approaches for avoiding such problems, a chimeric antibody has been developed (Patent Documents 1 and 2). The chimeric antibody contains parts of antibodies derived from two or more species (mouse antibody variable region, human antibody constant region and the like). An advantageous point of such a chimeric antibody is that it keeps the characteristics of mouse antibody but can stimulate human complement or human immune cells since it has human Fc. However, it is known that such a chimeric antibody still induces “human anti-chimeric antibody” namely “HACA” response (Non-patent Document 7).
Further, it has been developed a recombinant antibody in which only a complementarity determining regions (“CDRs”) of a part of an antibody were substituted (Patent Documents 3 and 4). By the use of a CDR grafting technique, an antibody comprising mouse CDR and human variable region framework and human constant region, so-called “humanized antibody” (Non-patent Document 8). Further, by the use of a human antibody producing mouse or by a screening using a human antibody library, broadly utilized techniques have been provided also regarding preparation of complete human antibodies (Non-patent Documents 9 and 10).
Regarding IL-3Rα:
IL-3Rα is the α chain of IL-3 receptor, belongs to a cytokine receptor family and shows weak affinity for IL-3 as its ligand. By forming a hetero receptor with its β chain (CD131, hereinafter also referred to as IL-3Rβ), an IL-3 receptor has a strong binding and transfers a signal such as growth, differentiation and the like into a cell through intracellular region of the β chain. IL-5 receptor α chain and GM-CSF receptor α chain share the β chain in common.
IL-3Rα is a type I membrane protein of single-pass transmembrane, and it is known based on the sequence that an IL-3 binding site and a fibronectin type III site are present in the extramembrane region. It is known that there is no structure which can transfer a signal in the intramembrane region. Though three-dimensional structure of IL-3Rα has not been analyzed yet, it can be assumed that structures of cytokine receptors are similar between families since position of cysteine residue which forms the structurally important S—S bond is preserved in most cases. Among the same cytokine receptors, crystalline structures of IL-13 receptor α chain, IL-4 receptor α chain and GM-CSF receptor α chain have been analyzed. Based on the information of these cytokine receptor families, it can be assumed that the extramembrane region of IL-3Rα is roughly divided into 3 domains (A-B-C domains). It is known that an antibody 7G3 which recognizes A domain of human IL-3Rα blocks IL-3 signaling (Non-patent Document 11). In addition, expression of an A domain-deficient IL-3Rα molecule has been reported (Non-patent Document 12), and as a matter of course, an antibody which recognizes A domain cannot recognize A domain-deficient IL-3Rα. In addition, it is considered that C domain is the root of IL-3Rα molecule and has a high possibility to three-dimensionally inhibit association of IL-3Rβ with IL-3Rα.
IL-3 is the only a ligand which is known as a ligand of IL-3Rα. IL-3 is a hematopoietic factor which is known to accelerate colony formation of the following: erythrocyte, megakaryocyte, neutrophil, eosinophil, basophil, mast cell and a monocyte system cell. It is known that IL-3 also stimulates a precursor cell having pluripotency, but IL-3 is rather said to accelerate a differentiation of not an immature stem cell having autonomous replication ability but a precursor cell committed to differentiation.
It is known that IL-3Rα relates to the growth and differentiation of myeloid cells by forming a heterodimer with β chain and thereby transferring the IL-3 signaling into the cell via the Serine/Threonine phosphorylation pathway. It is known that IL-3Rα is expressed in Granulocyte-Macrophage Progenitor (GMP) or Common Myeloid Progenitor (CMP) among hematopoietic precursor cells and induces growth and differentiation into neutrophil and macrophage systems via the IL-3 signaling. On the other hand, it has been reported that the Megakaryocyte Erythroid Progenitor (MEP) presenting in the downstream of CMP does not express IL-3Rα different from the GMP which is also present in the downstream.
Regarding the AML stem cell, Bonnet and Dick have reported that the AML stem cell is present in the CD34 positive CD38 negative fraction (Non-patent reference 13). Further, by comparing with the same fraction (CD34 positive CD38 negative) of normal stem cell, Jordan et al. have found that IL-3Rα is highly expressed in the AML stem cell (Non-patent reference 14). A high potential of IL-3Rα as a marker of not only AML stem cell but also leukemia stem cell has also been reported in the plural of reports thereafter (Non-patent references 15 and 16). In the treatment of cancers including leukemia, it is important that only the cancer cells are removed without injuring normal cells as many as possible, and it is considered that this difference in the expression of IL-3Rα between normal stem cell and leukemia stem cell is useful in the treatment targeting at the leukemia stem cell.
Regarding IL-3Rβ which forms a heterodimer with IL-3Rα, there is no report that IL-3Rβ is highly expressed leukemia stem cell, and also in the case of a microarray in which expression of mRNA in leukemia stem cell and normal stem cell is compared in fact, IL-3Rβ is not identified as a molecule in which its expression is increased in leukemia stem cell (Non-patent reference 17).
Regarding IL-3Rβ which forms a heterodimer with IL-3Rα, there is no report that IL-3Rβ is highly expressed leukemia stem cell, and also in the case of a microarray in which expression of mRNA in leukemia stem cell and normal stem cell is compared in fact, IL-3Rβ is not identified as a molecule of which expression is increased in leukemia stem cell (Non-patent reference 18).
The presence of a leukemia cell which depends on IL-3 has been known for a long time, and the old studies are studies focused on a blast cell which occupies most of the leukemia cells. According to the recent studies on leukemia stem cell, it is said that the leukemia stem cell acquires antitumor agent resistance by exhaustively suppressing its growth. In addition, it is considered that an IL-3 reactive blast cell has high proliferation ability so that it is assumed that such a cell is effective in the general treatment using an antitumor agent.
As a candidate of the agent targeting at an IL-3R receptor, the IL-3 itself was administered for a long time to patients of hematopoietic insufficiency but it did not become a drug as a result. A clinical trial for a fusion protein in which diphtheria toxin is added to IL-3 is in progress aiming leukemia as a target of the disease. Regarding the IL-3 and diphtheria toxin-IL-3 fusion, these are not suitable as the agents which are targeting at cells in which expression of IL-3Rα is specifically increased, since IL-3 binds strongly not a protein of IL-3Rα alone but a hetero protein of IL-3Rα and β due to properties of IL-3. On the other hand, as a candidate of an agent targeting at IL-3Rα, a first phase result of an IL-3Rα human mouse chimeric antibody 7G3 has been reported (Non-patent Document 19). Since the 7G3 chimeric antibody uses for the purpose of blocking of IL-3 signaling as the mechanism of AML therapy, this is not an agent aimed at removing IL-3Rα positive cells. Also, although some other IL-3Rα antibodies are known (9F5 (Becton Dickinson), 6H6 (SANTA CRUZ BIOTECHNOLOGY) and AC145 (Miltenyi-Biotech)), these do not have the ability to remove the cells highly expressing IL-3Rα.